We seek to extend promising studies showing that prostate specific antigen (PSA) and glandular kallikrein (hK2) occur in several forms in blood of prostate cancer (CaP) patients and that measurement of the proportion of PSA in the form of PSAalpha2-macroglobulin (alpha2M) improves the discrimination between early CaP and benign prostatic hyperplasia (BPH). This will be confirmed with well-characterized prospective patient samples. Moreover, we will measure the proportion of PSA in its various forms in these patients, and identify and measure forms of the related novel CaP marker, hK2. We found that hK2 exists in several forms in CaP plasmas, including hK2-alpha2M. Clinical assays do not detect PSA-alpha2M and there are no clinical assays for hK2. We will determine which combination of measurements provides the greatest discrimination between CaP and BPH. Since forms of PSA also vary in early vs. advanced CaP, our assays may also be useful in prognosis. The goal is to provide simple ELISA assays that will reduce the invasive, expensive procedures that are needed for diagnosis and ELISAs that are prognostic, to help physicians choose how aggressively to treat CaP patients, or to identify BPH patients who will develop CaP later. It is hypothesized that PSA and hK2 are secreted as zymogens, activated when needed, and then inactivated by protease inhibitors. The pathophysiology of cells that synthesize PSA and hK2 vary in CaP vs. BPH and possibly in different courses of CaP. The time between PSA or hK2 secretion and translocation to the blood may differ, and the enzymes and inhibitors to which they are exposed in the prostate differs from that in blood. Thus, a different spectrum of PSA and hK2 forms will be found in CaP compared to BPH, and in different courses of CaP. To meet our goals, we will: 1. Identify forms of hK2 in CaP and BPH plasmas and develop assays for relevant hK2 forms. Purify hK2 and prepare antibodies and standard hK2-inhibitor complexes for relevant hK2 forms. 2. Prospectively collect serial blood samples from 300 CaP patients and document full histories. Collect prospective samples from 300 undiagnosed patients with elevated PSA. 3. Measure PSA-alpha2M PSA-ACT, free PSA and "total" PSA in each sample and calculate the proportions of each PSA form. Similarly measure relevant hK2 forms. 4. Correlate the proportions of various forms of PSA and hK2 to the course of disease in all CaP patients to determine the prognostic value of each measurement. 5. Correlate the proportions of various forms of PSA and hK2 to the subsequent diagnosis of CaP or BPH in the patients who were undiagnosed. Determine the specificity and sensitivity of each measurement to determine its diagnostic value. Techniques will include protein purification, antibody and ELISA development, immunoblotting, enzyme activity assays, and peptide synthesis.